Disorganization (Ds) is a single gene mutation that has profound effects on mouse development. Unlike most mutations where particular traits with diagnostic features are inherited among generations, the heritable trait in mice with the Ds mutation is the propensity to make birth defects in an unpredictable manner - no two mice are affected in identical ways. Affected mice show agenesis, malformation, or duplication of structures and organs. All anatomical features are prone to malformation. Remarkably, these mice are not prone to cancer, spotting, behavioral, longevity or fertility problems. We previously showed that Ds is one of the few examples in mammals of a true dominant, gain-of-function (or dominant negative) mutation. Determining the identity of the Ds gene is important because deep insights into the regulation of pattern formation during development can be gained. In addition, we may gain insights into the molecular nature of mutations that show variable expression and low penetrance that are characteristic of many birth defects in humans. We mapped Ds to a 0.2 cM (about 600 kb) segment of mouse Chr 14, cloned in two BAC contigs (129/Sv and C57BL/6J), obtained about 260 kb of wild-type genomic sequence, identified about 20 candidate genes, and eliminated two genes by sequence analysis, seven genes by recombination, and most of the exons of four other candidate genes as the site of the Ds mutation. Based on these results, we now propose two Specific Aims: Specific Aim 1: To identify candidate Ds mutations by completing the genomic sequence of the Ds locus from Ds/Ds mutant homozygotes and comparing this to the wild-type sequence. Specific Aim 2: To test candidate DNA sequence differences in hit-and-run engineered mutant mice.